The expression of functional antibodies and antibody fragments in E. coli is known in the prior art, but these methods require the use of signal sequences which direct polypeptide transport into the periplasm. When expression takes place in the E. coli periplasm, the expression yields are in the range of a few xcexcg per liter of culture medium (Ayala et al., Bio Techniques 13, pp. 790-799, 1992). In addition, refolding experiments are often required in order to obtain functionally active antibody fragments (such as Fab) or antigen-binding regions (such as a single chain Fv(sFv)). There is a need therefore, to develop improved methods for expressing functionally active antibodies and antibody fragments. The prior art does not teach recombinant production of antibodies or antibody fragments which can be isolated from the cytoplasm in functional form. Such molecules would be useful in the production of therapeutic agents.
Bagshawe describes a method for generating cytotoxic agents that are directed towards cancer sites, termed Antibody Directed Enzyme Prodrug Therapy (ADEPT). Bagshawe, Br. J. Cancer, vol. 60, pp. 275-281, 1989. Using ADEPT, an antibody or antibody fragment that specifically binds to a cancer cell is fused to an enzyme that is capable of converting a non-toxic drug into a toxic drug. Only those cells to which the fusion protein is bound will be killed upon administration of the precursor of the toxic drug.
The xcex2-glucuronidase of Escherichia coli has been well characterized biochemically and genetically. The gene (uid A) has been cloned by Jefferson et al. (PNAS vol. 83, pp. 8447-8451, 1986) and employed as a reporter gene for heterologous control regions.
xcex2-Glucuronidase (xcex2-D-glucuronoside glucuronosohydrolase, E.C. 3.2.1.31) is an acid hydrolase which catalyzes the cleavage of xcex2-glucuronides. As a result of the mammalian glucuronidases having been intensively investigated, a variety of substances are available for histological, spectrophotometric and fluorometric analyses. This enzyme has gained new, additional importance in its use for fusion proteins for targeted tumor therapy. In this connection, human glucuronidase is used in the form of a fusion protein which contains antibodies/antibody fragments or antigen-binding regions (Bosslet et al., Br. J, Cancer, 65, 234-238, 1992). As an alternative to the human enzyme, it is also possible to use the homologous E. coli xcex2-glucuronidase. One of the advantages of the E. coli xcex2-glucuronidase is that its catalytic activity at physiological pH is significantly higher than that of the human xcex2-glucuronidase.
In the past, it has only been possible to express antibody fragment-enzyme fusion molecules periplasmically in E. coli. The enzyme moiety which is used in this context is therefore always composed of periplasmic E. coli enzymes such as xcex2-lactamase (Goshorn et al., Canc. Res. 53, 2123-2117, 1993).
An E. coli strain which is deficient in thioredoxin reductase (TRR), for example the strain AD 494, is capable of forming disulfide bridges in the cytoplasm and thus enzymes which are naturally secretory, for example alkaline phosphatase, can be expressed intracellularly See Derman et al., Science, 262:1744-1747, 1993. Derman describes the selection and isolation of TRR-deficient E. coli mutants.
The prior art does not teach expression of an antibody fragment-enzyme fusion molecule using a cytoplasmic E. coli enzyme, such as xcex2-glucuronidase, which is functionally activexe2x80x94i.e., which retains both enzymatic activity and antigen-binding ability of the antibody moiety. As a rule, functionally active expression of most antibodies or antibody fragment molecules requires defined signal sequences for exporting the expressed molecules via the endoplasmic reticulum into the culture medium (animal cells and yeast) or into the periplasm (E. coli). It is only in the endoplasmic reticulum or in the periplasm that the necessary oxidative conditions pertain for forming the disulfide bridges which are important for functional activity. In addition, the secretory synthesis route is often crucial for the correct three-dimensional folding of the expressed protein.
Thus, it is an object of the invention to provide a method for the production of functional antibodies and antibody fragments in E. coli in which the antigen-binding polypeptides can be isolated from the cytoplasm without the need for further processing such as protein folding and disulfide bond formation.
It is also an object of the invention to provide a method for the production of fusion polypeptides in E. coli comprising antibody or antibody fragment and an enzyme, in which the antibody or antibody fragment and the enzyme retain functionality and in which the functional fusion polypeptide can be isolated from the cytoplasm without the need for further processing such as protein folding and disulfide bond formation.
The invention relates, therefore, to processes for the recombinant expression of antibodies, antibody fragments or antibody fragment fusion molecules containing cytoplasmic mammalian or E. coli enzymes as fusion partners using thioredoxin reductase-deficient E. coli strains and subsequent isolation of the expression products from the cytoplasm.
The invention relates to the cytoplasmic expression of antibodies, antibody fragments and antibody fragment fusion molecules in E. coli. In particular, antibody fragment fusion molecules having an antibody moiety which is directed against tumors and an enzyme moiety which cleaves a nontoxic prodrug to give the toxic drug can be advantageously prepared in this way while retaining their respective functional properties.
Accordingly in one embodiment, the invention provides a method for producing an antibody or antibody fragment comprising:
a) transforming a thioredoxin reductase-deficient E. coli strain with a nucleotide molecule encoding said antibody or antibody fragment;
b) culturing said transformed E.coli strain to allow for expression of said antibody or antibody fragment; and
c) isolating said antibody or antibody fragment from the cytoplasm of said transformed E.coli. 
In a further embodiment, the invention also provides a method for producing a fusion protein comprising an antibody or antibody fragment and an enzyme, said method comprising:
a) transforming a thioredoxin reductase-deficient E. coli strain with a nucleotide molecule encoding said fusion protein;
b) culturing said transformed E.coli strain to allow for expression of said fusion polypeptide; and
c) isolating said fusion polypeptide from the cytoplasm of said transformed E.coli. 
In another embodiment, the antibody fragment used in the methods of the invention is selected from the group consisting of an Fab fragment, an Fv fragment, an sFv fragment and an F(abxe2x80x2)2 fragment. The antibody used in the methods of the invention can be a humanized antibody. The invention further provides an embodiment wherein the antibodies used in the method of the invention can be antibodies or antibody fragment binds specifically to tumor cells.
In another embodiment, an enzyme used in the method for making a fusion protein is capable of cleaving a nontoxic prodrug to produce a toxic drug and may be a human cytoplasmic enzyme.
In a further embodiment, the fusion protein produced according to the invention comprises an antibody or antibody fragments that is capable of specifically binding to tumor cells and an enzyme capable of cleaving a nontoxic prodrug to produce a toxic drug.
In a further embodiment, the fusion protein produced according to the invention comprises a humanized antibody and a human cytoplasmic enzyme. In another embodiment, the invention comprises a fusion protein comprising E. coli xcex2-glucuronidase.
In another embodiment, the invention provides fusion proteins comprising an antibody or antibody fragment and an enzyme. The invention further provides fusion proteins produced according to the methods of the invention. In a further embodiment, a fusion polypeptide comprising E. coli xcex2-glucuronidase and an antibody or antibody fragment is provided.
In a further embodiment, a nucleotide sequence encoding a fusion protein comprising an antibody or antibody fragment and an enzyme is provided. In still a further embodiment, the invention provides a nucleotide sequence encoding a fusion polypeptide comprising E. coli xcex2-glucuronidase and an antibody or antibody fragment. In yet another embodiment, a nucleotide sequence encoding the amino acid sequence in FIG. 5 which begins at nucleotide number 666 and ends at nucleotide number 3162 is provided. In a final embodiment, a nucleotide sequence is provided wherein said sequence is the nucleotide sequence in FIG. 5 which beings at nucleotide number 666 and ends at nucleotide number 3165.